Acquisition of host-derived carbon in biomass of the ectomycorrhizal fungus Pisolithus microcarpus is correlated to fungal carbon demand and plant defences

Abstract Ectomycorrhizal (ECM) fungi are key players in forest carbon (C) sequestration, receiving a substantial proportion of photosynthetic C from their forest tree hosts in exchange for plant growth-limiting soil nutrients. However, it remains unknown whether the fungus or plant controls the quantum of C in this exchange, nor what mechanisms are involved. Here, we aimed to identify physiological and genetic properties of both partners that influence ECM C transfer. Using a microcosm system, stable isotope tracing, and transcriptomics, we quantified plant-to-fungus C transfer between the host plant Eucalyptus grandis and nine isolates of the ECM fungus Pisolithus microcarpus that range in their mycorrhization potential and investigated fungal growth characteristics and plant and fungal genes that correlated with C acquisition. We found that C acquisition by P. microcarpus correlated positively with both fungal biomass production and the expression of a subset of fungal C metabolism genes. In the plant, C transfer was not positively correlated to the number of colonized root tips, but rather to the expression of defence- and stress-related genes. These findings suggest that C acquisition by ECM fungi involves individual fungal demand for C and defence responses of the host against C drain.


Introduction
Ectomycorrhizal (ECM) fungi colonize the roots of most trees in temperate and boreal forests and, as these ecosystems make up the majority of the global terrestrial carbon (C) sink, are crucial to C cycling (Taylor et al. 2000, Martin et al. 2001, Pan et al. 2011, Averill et al. 2014, Wu et al. 2022. In nutrient-limited forests, ECM fungi colonize rece pti ve plant roots and provide plants with nutrients fr om surr ounding soil. These nutrients ar e r eleased into a specialized colonization structure in the root tip called the Hartig net, where ECM fungi obtain up to 30% of photosynthetically fixed plant C (Leake et al. 2004, Hobbie 2006. While ECM fungi have specialized abilities to acquire plant growth-limiting nutrients such as nitrogen (N) and phosphorus (P) from soil organic and inorganic matter, and improve water uptake, the y remain de pendent on root-exuded C due to their limited capacities to metabolize complex carbohydrates (Nehls et al. 2010, Lindahl and Tunlid 2015, Liu et al. 2020. The direct access of ECM fungi to this C entails a competitive adv anta ge ov er other soilborne microbes and therefore alters soil r espir ation and may impr ov e belowgr ound C stor a ge (Gadg il and Gadg il 1971, 1975, Averill et al. 2014, Averill and Hawkes 2016, Gorka et al. 2019.
Despite the importance of ECM symbiosis in C cycling, mechanisms controlling host-to-fungus C transfer and greater ecosystem impacts of ECM-associated C cycling are not well understood (r e vie wed in Stuart and Plett 2020 ). Some studies suggest that ECM host plants control C allocation through reciprocal rew ar ds-or sanctions-based mec hanisms, wher eby plants r e w ar d more C to fungi that provide more growth-limiting nutrients (Kytöviita 2005, Kiers et al. 2011, Casieri et al. 2013, Bogar et al. 2019. Alternativ el y, plants may incr ease belowgr ound C allocation in r esponse to soil nutrient limitation to encour a ge gr eater soil nutrient supply by the fungi (Hobbie 2006, Nehls et al. 2010, Näsholm et al. 2013. Others suggest that belowground C allocation may operate based on a source-sink mechanism where nutrient transfer is influenced by fungal C demand. This sink may be created by increases to fungal biomass production or allocation of C to w ar ds assimilating N (Wallander 1995, Corrêa et al. 2008, Lemoine et al. 2013. Mor eov er, host defence r esponses ma y pla y a role in controlling C loss via attack on, and senescence of, roots colonized by uncooper ativ e fungi (Kiers and Denison 2008, Garcia et al. 2015, Hortal et al. 2017, Bogar et al. 2019. Mor e r esearc h is needed to understand these defence mechanisms. Understanding of ECM C transfer at the molecular level is limited (Durall et al. 1994, Pumpanen et al. 2009, Heinonsalo et al. 2010, Pickles et al. 2016, although transporters and enzymes that may facilitate sugar transfer and catabolism at the plant-fungal interface have been found to be upregulated in colonized plant roots (Wright et al. 2000, Nehls et al. 2010, Plett et al. 2015a, b , Bouffaud et al. 2020, Ruytinx et al. 2021, Tang et al. 2021. While some host-derived C may be lost via ECM fungal r espir ation or exudation, a substantial amount is sequestered by rapid conversion into various storage compounds including trehalose, mannitol, and glycogen (Martin et al. 1987, López et al. 2007, Nehls et al. 2007, Hagenbo et al. 2019. Such a sequestration mechanism suggests that the ability of ECM fungi to produce biomass should be correlated to their acquisition of host-deri ved C. Leak e et al. ( 2001 ) noted that increases in the hyphal density of Paxillus involutus occurred in litter patches that contained greater amounts of C derived from the host plant Pinus sylvestris , indicating a link between fungal biomass production and C acquisition. Ho w e v er, ther e is a curr ent lac k of studies quantifying the correlation between fungal biomass production and amounts of host-deriv ed C acquir ed by a gr eater r ange of ECM fungi, partl y due to the difficulties of measuring fungal biomass and C allocation in field experiments (Hobbie andAr ger er 2010 , Wallander et al. 2013 ).
Ther e is consider able genetic div ersity amongst ECM fungal species leading to potential interspecies variation in the genetic mechanisms supporting C acquisition from their hosts (Read and Per ez-Mor eno 2003, Rinaldi et al. 2008, Tedersoo and Smith 2013, Martin et al. 2016, Tedersoo and Brundrett 2017. For example, C starv ation, r ather than C av ailability, w as sho wn to induce expression of fungal monosaccharide transporter genes by Laccaria bicolor (Fajardo López et al. 2008 ). In Amanita muscaria , the sugar transporter AmMst1 is upregulated under increased C sugar concentrations (Nehls et al. 2001 ). While these differ ences in C exc hange and metabolism hav e been found to occur between distantly related fungal species, more recent work would suggest that there can be distinct variation within one ECM fungal species (Hortal et al. 2017, Plett et al. 2021. This suggests that understanding of this function should be impr ov ed to a finer scale than current genus-level knowledge.
Here , we in vestigated the effects of intraspecies transcriptomic and physiological variability on C exchange between nine isolates of Pisolithus microcarpus that ranged in mycorrhization potential, and their host Eucalyptus grandis . The amount of C transferr ed fr om host to fungus and incor por ated into fungal biomass was measured via 13 C stable isotope labelling using a microcosm system, and gr owth differ ences betw een the fungal isolates w ere examined to determine whether gr owth-r elated tr aits corr elated with the amounts of C acquir ed. Gene expr ession in the fungi and in the host plant wer e anal ysed to find expression patterns that may correlate to C acquisition by the fungi from the plant. Based on the liter atur e discussed abo ve , we hypothesized that there would be significant intraspecies variation in the amounts of host-derived C in fungal biomass, and that these amounts would positiv el y corr elate with fungal growth and colonization rate. We also hypothesized that the expressions of both plant and fungal genes relating to C transport and metabolism, including both catabolism and biosynthesis, would correlate with amounts of host-derived C, and that decreased gene expression of plant defence mechanisms would allow for greater C acquisition by the fungi.

Biological material and experimental microcosm setup
Nine P. microcarpus isolates were originally isolated from various locations across Australia (scientific license number S13146; Supplementary Table S1; Keniry 2015 ) or New Caledonia (Jourand et al. 2010 ). The fungal isolates were selected as they represent a gradient of host mycorrhization potential (e.g. Plett et al. 2015a ). Pur e cultur es of these fungal isolates wer e maintained on modified Melin Norkrans (MMN) agar plates (0.5 g/L (NH 4 ) 2 HPO 4 , 0.3 g/L KH 2 PO 4 , 0.14 g/L MgSO 4 .7H 2 O, 10 g/L glucose, 1 mL/L of CaCl 2 5% w/v stock solution, 1 mL/L of NaCl 2.5% w/v stock solution, 1 mL/L of ZnSO 4 0.3% w/v stock solution, 133 μL/L of thiamine 0.1% w/v stock solution, 1 mL/L of citric acid + Fe EDTA 1.25% w/v stock solution and 13 g/L agar in de-ionized water, pH 5.5). The plates were incubated in a growth chamber in the dark at 25 • C (ICP 800, Memmert, Sc hwabac h, Bav aria, German y).
Phylogenetic analysis of the P. microcarpus isolates was conducted with the online tool Phylogeny.fr (Dereeper et al. 2008 ). ITS sequences were obtained from the National Center for Biotechnology Information (NCBI) Nucleotide database ( https://www.ncbi .nlm.nih.gov/nuccore ) or sequenced at the Hawkesbury Institute for the Environment (Western Sydney Univ ersity, Ric hmond, Ne w South Wales, Australia; Supplementary Table S2). ITS sequences were aligned using MUSCLE 3.8.31 and poorly aligned positions and div er gent r egions wer e eliminated using Gbloc ks 0.91b. The phylogenetic tree was constructed using the maximum likelihood method (PhyML3.1/3.0 aLRT) and drawn with TreeDyn 198.3.
Eucalyptus grandis seeds (seedlot no. 21 068) were obtained from the Commonwealth Scientific and Industrial Researc h Or ganisation (CSIRO, Clayton, Victoria, A ustralia) A ustr alian Tr ee Seed Centr e. Eucal yptus grandis seeds were surface sterilized with 30% hydr ogen per oxide for 10 min and germinated on 1% w/v water agar in a plant growth chamber (TRISL-495-1-SD, Thermoline Scientific, Smithfield, New South Wales, Australia) under a 16-h light cycle at 25 • C. After 4 weeks of growth, the seedlings wer e tr ansferr ed, thr ee seedlings per plate and on top of a sterile cellophane membrane, to fresh, half-strength ( 1 2 ) MMN agar plates containing 0.1% w/v glucose . T he plates were incubated for another 4 weeks under the same conditions . Meanwhile , small squares (0.5 × 0.5 cm) of each P. microcarpus isolate were cut from the growing edge of the fungal cultures grown on 1 × MMN cultur es, and wer e tr ansferr ed to fr esh 'low glucose' 1 2 MMN a gar plates, made with the same r ecipe as 1 2 MMN but onl y containing 0.01% w/v glucose, on top of a sterile cellophane membrane. Eight cultures per isolate were prepared. The plates were incubated in a growth chamber (ICP 800) in the dark at 25ºC.
After 2 weeks of fungal growth on the low glucose 1 2 MMN, individual E. grandis seedlings were carefully transferred onto the edge of growing P. microcarpus mycelium. In total, five plant + fungus microcosms ('ECM' condition) per P. microcarpus isolate were set up. As contr ols, thr ee micr ocosms per isolate with fungi onl y [fr eeli ving m ycelium (FLM) contr ol] wer e also maintained, and thr ee E. grandis seedlings were transferred onto low glucose 1 2 MMN as plant only controls . T he plates were sealed with one-third micropor e ta pe at the top of the plate, to allow for fr ee gas exc hange, and tw o-thir ds electrician's tape . T he plates were placed in a plant gr owth c hamber (GC20 BDAF, Bio Chambers Incor por ated, Winnipeg, Manitoba, Canada) at a 45 • C angle and incubated under a 16 h photoperiod, light intensity of 500 μmol m −2 s −1 , 25 • C/18 • C day/night temper atur e, 70% r elativ e humidity, and ambient CO 2 (400 ppm) for 2 weeks, at which the fungus would hav e full y colonized the root system and established nutrient trading as previously shown (Hortal et al. 2017 ). Within the 2 week co-culture, at 9 days post fungal contact, two holes were opened on the front of each plate using a soldering iron and the holes were covered with micr opor e ta pe to further incr ease gas permeability into the plates . T he plates were placed in a 175 L airtight polycarbonate chamber with an air circulation fan and a gas injection port inside the plant growth chamber (for more details, see Hortal et al. 2017 ). The tank was injected with 12 mL of 99 atom % 13 CO 2 (Sigma-Aldrich, St Louis, MI, USA) and the plates were left in the tank for 5 hours under circulating air. Following this, the tank was opened and allo w ed to equilibrate back to ambient levels of 13 CO 2 . At day 11 of co-culture, the microcosms were once again placed in the labelling chamber and another 12 mL of 99 atom % 13 CO 2 was injected into the tank and the plates were again left in the tank for 5 hours under circulating air followed by return to ambient 13 CO 2 conditions until harv est. Fr ee-li ving m ycelium and plant only contr ol plates wer e gr own and incubated under the same conditions.
For the measurement of fungal biomass, four to fiv e mor e r eplicates per isolate of the FLM control plates were set up and treated in the same way as described abo ve , exce pt that the y did not undergo 13 C labelling.

C stable isotope analysis
T hree da ys after the second injection of 13 CO 2 , following sampling for RNA-seq analysis (see below), all of the remaining fungal extr a-r adical mycelium (ERM) and plant leav es fr om the low glucose 1 2 MMN plates (four to five replicates of the ECM symbiosis plates and thr ee r eplicates of the FLM fungus-only plates) were harv ested, ov en-dried at 40ºC and ground to a fine powder. 13 C atom % values were measured by running about 1 mg of ground fungal tissue or about 2 mg of ground plant tissue on an elemental analyser and isotope ratio mass spectrometer (UC Davis Stable Isotope Facility, Da vis , C A, USA). Outlier 13 C atom % values were identified using the interquartile range test and r emov ed from the data set. 13 C atom % values were converted to amounts of C tr ansferr ed fr om the plant host to the fungus via symbiosis and retained, as respired or exuded C was not accounted for, using the calculations below based on methods pr e viousl y described (Tomm et al. 1994, He et al. 2009. Herein, 'C acquisition data' refer to these C values.
where %C symbiosis is the percentage of fungal C derived from the plant host, 13 C ERM is the 13 C atom % of the fungal ERM of the symbiosis plates, 13 C FLM is the av er a ge 13 C atom % of the fungal mycelium of the fungus only plates, 13 C leaf is the 13 C atom % of the leaves of the symbiosis plates, C symbiosis is the amount of fungal C derived from the plant host disr egarding an y subsequentl y r espir ed or exuded C, %C fungal is the av er a ge percenta ge of total C in the dried fungal mycelium, and fungal biomass is the av er a ge mass of the dried fungal mycelium of the fungus only plates.

Fungal growth parameters
The following parameters were measured as they can be considered as proxies for fungal growth. Colonization percentage was calculated from four to five replicates per fungal isolate from the ECM symbiosis plates . T his calculation was the number of lateral plant root tips colonized by the fungus (i.e. exhibiting short, thickened appearance and the pres-ence of a fungal mantle over the root) divided by the total lateral plant root tips in contact with fungal mycelium.
For the measurement of Hartig net depth and mantle thickness, colonized plant root tips from each isolate treatment wer e harv ested fr om the ECM symbiosis plates, fixed in 4% w/v paraformaldehyde and stored at 4ºC for at least 24 h. The root tips were then embedded in 6% w/v agarose and refrigerated ov ernight. For eac h tr eatment, cr oss-sections of 30 μm thickness fr om thr ee independent biological r eplicates wer e cut using a Leica VT1200 vibratome (Leica Microsystems, Mt Waverley, Victoria, Australia), stained with DAPI stain and imaged using an inverted Leica SP6 confocal microscope (Leica Microsystems). The Hartig net depths and thicknesses of the fungal mantles surrounding the plant root tips were measured on four to seven sections per crosssection using the Ima geJ pr ocessing pr ogr am (National Institutes of Health, Bethesda, MD, USA; Schneider et al. 2012 ).
Gr owth r ate , biomass , and hyphal density of the fungal isolates were determined as follows from four to five replicates per isolate of the fungus only plates, rather than from symbiosis plates, due to the difficulty in fully separating fungal hyphae from plant roots. After 2 and 4 weeks of growth on the low glucose media, the perimeters of the fungal colonies were marked, the colonies wer e ima ged and ar eas of the fungal colonies and of the original a gar bloc ks determined using Ima geJ . The mycelium was harv ested, ov en-dried at 40ºC and weighed to determine the biomass v alues. Av er a ge r adial mycelial gr owth, calculated as the ar ea of r adial gr owth per day fr om the second week of gr owth, or 'plant contact', to the fourth week of growth, or 'harvest', was used as a proxy for growth rate during symbiosis with the plant host. Hyphal density was calculated as the fungal biomass per unit area of the fungal colony at harvest.

RN A extr action and RN A-seq anal ysis
After 4 weeks of growth on the low glucose 1 2 MMN, a strip of fungal ERM (including the oldest and newest part of the colony) and plant roots in contact with the fungus (excluding tap roots) were harv ested fr om thr ee r eplicates of symbiosis or plant onl y contr ol low glucose plates and immediately frozen at −80 • C. Total RNA (from the combined fungal ERM and plant roots for three replicates per treatment) was extracted using the ISOLATE II miRNA kit (Bioline , London, UK). T he lar ge RNA fr action fr om these extractions was sequenced at the Joint Genome Institute (JGI) as detailed in Plett et al. ( 2020 ).
Prior to normalization of the count data, genes that were not expressed under any isolate condition (having an av er a ge r ead count within each condition of < 10 mapped reads for E. grandis and < 5 for P. microcarpus ) wer e r emov ed fr om the data. This r esulted in sets of 24 615 E. grandis transcripts and 12 959 P. microcarpus transcripts as 'expressed' under at least one condition. The DESeq2 R pac ka ge (v. 3.6.1) was used to normalize the count data. Functional annotations for the following genomes were obtained from the JGI

Hea tmap visualiza tion
Eucalyptus grandis C transporter genes, P. microcarpus C transporter genes, and P. microcarpus C metabolism genes were obtained from Hortal et al. ( 2017 ) and Plett et al. ( 2021 ). For heatmap visualization, fold change in gene expression for each isolate was cal- microcarpus . (A) ITS-based phylogenetic tree of the studied P. microcarpus isolates and the P. microcarpus r efer ence isolate 441 v 1.0. ITS sequences for P. albus , P. tinctorius , and Scleroderma citrinum are used as outgroups. Phylogenetic analysis and tree construction were performed with Phylogeny.fr using 'one click' mode. Branch length represents sequence div er gence (scale bar r epr esents 0.1 substitutions per site). (B) Pisolithus microcarpus isolates used in this manuscript grown on low glucose 1 2 modified Melin Norkrans agar plates. Each isolate is annotated in the figure as follows: culated by dividing the DESeq2-normalized gene expression values by the av er a ge expr ession v alues of all of the isolates (i.e. the baseline expression level), and log 2 -transformed. Heatmaps were generated using the online tool Morpheus (Broad Institute, Cambridge, MA, USA, https://softwar e.br oadinstitute.org/mor pheus ). Hier arc hical clustering for C transporter gene heatmaps was performed using the Euclidean distance measure. Carbon transporter and C metabolism genes of interest were identified as genes for which Wil3 had the highest or lo w est le v el of expr ession of the fungal isolates . T his is because Wil3 r eceiv ed significantl y gr eater amounts of 13 C from the host than most other fungal isolates, so genes with compar ativ el y higher or lo w er le v els of expr ession in Wil3 may be related to fungal C acquisition.

Pearson's correlation analysis of C acquisition data
The DESeq2-normalized count data for the expressed E. grandis and P. microcarpus transcripts were correlated to the amounts of host-deriv ed C acquir ed b y the fungi accor ding to the method described by Baute et al. ( 2015 ). Briefly, Pearson's correlation coefficients between the av er a ged C acquisition data and the count data, fr om the thr ee r eplicates on whic h tr anscriptomic analysis was performed, for eac h tr anscript wer e calculated. Tr anscripts with Pearson's correlation coefficients in the top percentile (q 0.99 , i.e. most positiv el y corr elated tr anscripts) and bottom percentile (q 0.01 , i.e. most negativ el y corr elated tr anscripts) of correlation values were identified. All Pearson's correlation coefficients for the identified transcripts were statistically significant according to the Pearson Correlation Table of Critical Values.
The functional annotations of these sets of positiv el y and negativ el y corr elated tr anscripts wer e searc hed to identify genes involved in C metabolism (biosynthesis and catabolism), defence/disease resistance, gro wth/cell c ycle regulation, host-fungus interaction, signal transduction, stress r esponse, tr anscription r egulation, or tr ansport, as these categories of genes may play a role in C acquisition beyond the C transporter and metabolism genes used for heatmap visualization.

Sta tistical anal yses
ANOVA and Tuk e y's Honest Significant Different (HSD) test for post-hoc anal ysis wer e performed on the C acquisition, gr owth, and colonization data using the R stats pac ka ge (v. 3.6.1). Principal component analysis (PCA) of log-transformed symbiotic C acquisition and fungal gr owth c har acteristics data was performed using the prcomp function in R. Correlation analyses were conducted in Microsoft Excel (v. 16) to examine the association between the amounts of host-derived C acquired by the fungal isolates and their growth parameters. For correlation between C acquisition and colonisation rate, data from paired samples were used. For correlation between C acquired and other growth parameters (biomass, hyphal density, Hartig net depth, mantle thickness, and growth rate), averaged C acquisition data and individual growth parameter data were used as these groups of datasets were obtained from nonpaired samples.

Gro wth char acteristics and acquisition of host-deri v ed C varies significantly between P. microcarpus isolates
Acquisition of host-derived C was investigated using nine fungal isolates. A phylogenetic tree was constructed and all isolates were found to group with P. microcarpus r efer ence isolate 441, distinct from the closely related P. albus and the more basal species P. tinctorius (Fig. 1 A). The morphology of the fungal colonies grown in vitro on low glucose plates varied notably, ranging fr om sparse, far-r eac hing mycelia to denser and shorter mycelia (Fig. 1 B). Symbiotic C acquisition by each P. microcarpus isolate from its host was determined through 13 C stable isotope analysis of the fungal ERM. Differences between the isolates in the percentage of ERM C deriv ed fr om symbiosis wer e not statisticall y significant, due to the large variation in the Wil4 measurements (Fig. 2 A). Statistical significance was observed when Wil4 was r emov ed fr om the data ( P < .01). While the percentage of m ycelial C deri v ed fr om symbiosis gives a proportional measure of host C present in ERM tissue per unit mass, it did not r epr esent the total amount of C estimated to be tr ansferr ed to fungal tissues . T her efor e, the total amount of host C acquired by symbiosis and retained in hyphal tissues (i.e. did not account for exuded or r espir ed C) was calculated using the C content and ov er all biomass estimates of the fungal colonies . T her e was significant v ariation in the amount of biomass produced by the isolates in vitro and in the percentage of C of the fungal mycelia (Table 1 ), amplifying the differences in C acquisition amongst the P. microcarpus isolates such that the amounts of host-acquired C differed significantly between the isolates (Fig. 2 B). Wil4, while containing a high percentage of C from symbiosis in some replicates, had the smallest amount of biomass of the fungi considered, and thus sho w ed the lo w est amount of total host C acquired. PCA was conducted to find relationships between symbiotic C acquisition by the fungus and measur ed fungal gr owth c har acteristics (Fig. 2 C). PCA 1 and 2 together explained 68.6% of the variance. Along the PC1 axis, the amount of symbiotic C acquired by the fungal isolates was most closely related to biomass, hyphal density and Hartig net depth.

Acquisition of host-deri v ed C by P. microcarpus is significantl y correla ted to biomass accumula tion, hyphal density, and hartig net depth
As predicted by the PCA, while fungal biomass did not correlate with the percentage of mycelial C derived from symbiosis, it did positiv el y corr elate with the amount of host-deriv ed C (Fig. 3 A and B). Similarly, the amount of host-derived C corr elated significantl y with the density of fungal hyphae (Fig. 3 C) and Hartig net depth (Fig. 3 D), but did not significantly correlate with the thickness of the fungal mantle surrounding plant root tips (Fig. 3 E) nor with radial mycelial growth rate (Fig. 3 F).

Root colonization rate negati v ely correlates with host C acquisition by P. microcarpus
Plant root colonization rate did not significantly differ between the fungal isolates used in this experiment (Fig. 4 A). Correlation anal yses wer e performed to determine whether plant root colonization rate correlated with the percentage or amount of hostderiv ed C acquir ed by the fungi. Colonization r ate was significantl y, negativ el y corr elated with the percenta ge of symbiotic C retained in the fungal mycelium ( P < .05; Fig. 4 B). Colonization rate had a weakly negative correlation with the amount of hostderived C, although this was not statistically significant ( P = .11; Fig. 4 C).

Pisolithus microcarpus C acquisition is more related to expression of C metabolism genes than C transporter genes
Plant and fungal gene expression in E. grandis roots colonized by each of the P. microcarpus isolates was considered and heatmaps of the expression of E. grandis C transporter and P. microcarpus C transporter and metabolism genes were gener-ated to investigate whether fungal C acquisition was related to plant and fungal C-related activity (Fig. 5 , Supplementary  Table S3).
Ov er all, ther e wer e no str ong tr ends between amounts of C acquired by the fungi and expression of plant C transporter genes (Fig. 5 A). Ho w e v er, when plant gene expression patterns were compared between the Wil3 treatment and all other isolates that r eceiv ed lo w er C from the host, 13 genes of putative interest were identified. Five plant genes were more highly expressed under the Wil3 treatment as opposed to all other isolates, and eight genes were repressed. The genes showing higher expression during symbiosis encoded major facilitator superfamil y tr ansporters ( Eucgr .B02021 , Eucgr .B02013 , Eucgr .H01571 ), an EgSWEET16 protein ( Eucgr.H04549 ), and a tonoplast monosacc haride tr ansporter ( Eucgr.I02208 ). The r epr essed genes encoded major facilitator superfamil y pr oteins ( Eucgr .B02309 , Eucgr .B02016 , Eucgr .H04438 , Eucgr .H04441 ), inositol transporters ( Eucgr .E03903 , Eucgr .E02426 ), a sugar transporter ( Eucgr.D00720 ), and a sucrose transporter ( Eucgr.F00464 ).
Similar to the E. grandis genes, the P. microcarpus C transporter genes also did not have strong trends with the C acquisition data (Fig. 5 B). Ho w e v er, ther e wer e gener al tr ends between C acquisition ability of a given isolate and gene expression of the following fungal major facilitator superfamil y tr ansporters (by protein ID): Pm113068, Pm579566, Pm171041, Pm678263, Pm676370, Pm677567, Pm654975, and Pm9181. Inter estingl y, the majority of these P. microcarpus C transporters had a negative trend with C acquisition, with the exception of Pm9181 and Pm579566. Fungal C metabolism genes with general trends with the C acquisition data were also identified (i.e. higher in Wil3 as opposed to other isolates; Fig. 5 C). These encoded the sugar/carbohydrate utilization enzymes fructokinase (Pm60756, Pm110989), phosphoglucose isomerase (Pm676950), and trehalase (Pm24996), and the carbohydrate biosynthesis enzymes galactinol synthase (Pm91096), glycogen synthase (Pm683186), and trehalose-6phosphate synthase (Pm677150). Inter estingl y, all of these C metabolism genes were repressed in isolates that received and retained higher host C with the exception of the trehalase gene Pm24996.

Pisolithus microcarpus C acquisition correlates with the plant expression of defence-and stress-related genes and regulatory genes
To further investigate the genes potentially involved in fungal C acquisition, a Pearson's corr elation anal ysis was conducted to determine the le v el of positiv e or negativ e corr elation between host gene expression and fungal C acquisition. The top and bottom percentiles of E. grandis tr anscripts corr elated with the amounts of C acquired by P. microcarpus fungi (i.e. the most positiv el y corr elated and most negativ el y corr elated tr anscripts, r espectiv el y) wer e consider ed. In total, ther e wer e 247 tr anscripts eac h in the top and bottom percentiles of correlations with the C acquisition data (q 0.99 Pearson = 0.52, q 0.01 Pearson = −0.50; Fig. 6 A, Supplementary Table S4).
As GO enrichment did not identify any specific pathways within these datasets, a search was conducted for genes involved in C acquisition beyond the C transporter and metabolism genes pr e viousl y identified. The categories of genes searched for are presented in Table 2 . 32% of all correlated genes (26% of positiv el y corr elated and 39% of negativ el y corr elated genes) r elated to growth/cell cycle and/or transcription regulation, including genes for methyltr ansfer ases, pentatricopeptide r epeat pr oteins, Figure 2. Pisolithus microcarpus shows significant intraspecies variation in acquisition of host-derived carbon (C) that is related to fungal biomass and hyphal density. (A) P er centage of mycelium C acquired by P. microcarpus isolates from E. grandis host. (B) Amount of 13 C acquired by P. microcarpus isolates from E. grandis host. (C) PCA showing variation between fungal isolates in terms of their amount of symbiotic C acquisition (black arrow) and fungal growth characteristics (coloured arrows). Different letters indicate statistically significant ( P < .05) differences as determined via the Tuk e y's HSD test. Error bars indicate standard error.

Isolate
Biomass (   tetr atricopeptide r epeat pr oteins, F-box superfamil y pr oteins, ubiquitin-associated proteins and zinc fingers ( Table 2 ). Genes related to defence/disease resistance and/or stress response accounted for 20% of correlated genes (22% of positiv el y corr elated and 19% of negativ el y corr elated genes), including genes for laccases, U-box superfamily proteins, and LRR domain and NB-ARC domain disease resistance genes. Carbon metabolism genes made up < 3% of all correlated genes. Correlation of amount of 13 C acquired from host with plant root colonization rate. Different letters indicate statistically significant ( P < .05) differences as determined via the Tuk e y's HSD test. Each dot represents the paired C and colonization data for one replicate. Dotted lines indicate lines of best fit.

Pisolithus microcarpus C acquisition negati v ely correlates with fungal expression of signalling, regulatory, and defence genes
Gene expression in the P. microcarpus isolates correlated to symbiotic C acquisition with E. grandis was also analysed via Pearson's corr elation anal ysis . T her e wer e 130 tr anscripts eac h in the top and bottom percentiles of correlations with the C acquisition data (q 0.99 Pearson = 0.67, q 0.01 Pearson = −0.58; Fig. 6 B, Supplementary Table S5). Similar to the analysis of E. grandis gene correlation, within this data set P. microcarpus genes relating to C acquisition were targeted (Table 3 ).
Only nine of the positively correlated genes fell into these categories, of which six were related to transcription regulation and none were related to C metabolism. Negatively correlated genes were largely annotated as being involved in signal transduction, tr anscription r egulation, gr o wth/cell c ycle regulation, and defence/disease resistance . T hree carbohydrate biosynthesis genes and se v en sugar/C catabolism genes were also identified as negativ el y corr elated, of whic h one was r elated to inositol metabolism.

Discussion
While knowledge of the factors that affect C acquisition by ECM fungi acr oss distantl y r elated linea ges hav e impr ov ed in r ecent years, understanding of how this is affected by genetic variability between closely related fungi is less well characterized. In this study, as hypothesized, the P. microcarpus isolates tested varied in the amounts of C acquired from E. grandis . This variation between the fungi allo w ed us to uncover physiological and genetic mechanisms that correlate with host-to-fungus C exchange. Earl y r eports considering C allocation to mycorrhizal root tips found that fine roots colonized by ECM fungi r eceiv ed between 5-18 × more photosynthate than uncolonized roots (Cairney et al. 1989, Wu et al. 2002. A significant portion of this C is then transferred into the ERM within the first few days (Wu et al. 2002 ). For this reason, it was originally hypothesized that isolates of P. microcarpus with higher amounts of host-derived C acquired would also show higher le v els of r oot tip colonization. Congruent with this theoretical framework, a study by Shinde et al. ( 2018 ) found that ECM-colonized Populus tremuloides plants under nonlimiting nutrient conditions had higher le v els of certain carbohydrates in Figure 5. Host-derived carbon (C) acquisition is lar gel y unr elated to expr ession of plant and fungal genes for C transport and metabolism. Heatmaps of DESeq2-normalized and log 2 -transformed expression values of (A) E. grandis C transporter genes, (B) P. microcarpus C transporter genes, and (C) P. microcarpus C metabolism genes. Heatmaps were generated using Morpheus online tool. Hierarchical clustering for C transporter gene heatmaps was performed using the Euclidean distance measure. Carbon metabolism genes were grouped by C metabolism pathway, as indicated to the right of the heatmap. Isolates labelled as 'high C' and 'low C' had statistically significant differences in amounts of acquired host-derived C, as determined by ANOVA. Arrows indicate genes of interest that are referenced in the text and are identified as genes for which Wil3 had the highest or lo w est level of expression out of the fungal isolates. Columns labelled as 'Ctrl' represent uninoculated control plants. Each additional column is ordered as in Fig.  2.2B based on r elativ e C ca ptur e fr om the plant and is labelled with the fungal isolate name as follows: W3-Wil3, PA-P. micro A, G5-G5, M3-MW3, M6-MD06, S9-SI9, R10-R10, R4-R4, and W4-Wil4. GLC, glucose, MFS, major facilitator superfamily. F igure 6. P earson's corr elation anal ysis r e v eals gene tr anscripts of E. grandis and P. microcarpus correlated with fungal carbon (C) acquisition. F requenc y histograms of Pearson's correlation coefficients for correlations between C acquisition data and transcriptomic data, and heatmaps of transcripts positiv el y (q 0.99 ) and negativ el y (q 0.01 ) correlated with C acquisition, for (A) E. grandis , and (B) P. microcarpus . Dotted lines in histograms indicate q 0.99 and q 0.01 of the Pearson's correlation coefficients (i.e. where the most positively and most negatively correlated genes are located, respectively). Heatmaps display fold change expression relative to the average expression of each gene across all conditions (DESeq2-normalized and log 2 -transformed). Hier arc hical clustering was performed using the Euclidean distance measure. Columns labelled as 'Ctrl' represent uninoculated control plants. Each additional column is ordered as in Fig. 2.2B based on relative C capture from the plant and is labelled with the fungal isolate name as follows: W3-Wil3, PA-P. micro A, G5-G5, M3-MW3, M6-MD06, S9-SI9, R10-R10, R4-R4, and W4-Wil4. Table 3. Predicted carbon (C)-related functions of P. microcarpus genes positiv el y and negativ el y corr elated with amount of C acquir ed from the host plant. Correlated genes were identified via Pearson's correlation analysis of DESeq2-normalized genes. their colonized roots, including starch and sucrose . Furthermore , plants colonized by Paxillus involutus , which had up to 13% higher colonization rates than L. bicolor , had higher levels of starch in their fine roots than L. bicolor -colonized plants. Ho w ever, the results of our current study demonstrate a negative correlation between C acquisition and colonization rates of the host root system. Findings by Hortal et al. ( 2017 ), who measured both colonization rate and symbiotic C content in the mycelia of three Pisolithus isolates associated with E. grandis , also found that colonization rate was not associated with host-derived C. Further studies examining both plant root colonization and C transfer from host to fungus are needed to gain a better understanding of the importance of root colonization rate in C acquisition and whether there are ECM fungal genera that require high rates of root colonization to obtain host C while others do not. Our aim to identify host genes putativ el y corr elated to gr eater C acquisition by an ECM fungus identified that 20% of genes correlated with this tr ait wer e involv ed in defence or stress responses. While plant defence and str ess r esponses to colonization by mycorrhizal fungi have been reported previously, they are often observed in the context of symbiosis formation or defence priming rather than the control of C transfer to symbionts (Pozo and Azcón-Aguilar 2007, Kiers et al. 2011, Garcia et al. 2015, Plett et al. 2015b, Kanekar et al. 2018, Watts-Williams et al. 2018, Dreischhoff et al. 2020. Ho w ever, plants are kno wn to employ defence mec hanisms a gainst r oot-dwelling fungi to limit r esource loss and to pr e v ent mycorrhizal fungi fr om becoming par asitic to w ar ds their hosts (Ferr eir a et al. 2007, Agr en et al. 2019. Supporting this, Hortal et al. ( 2017 ) found ele v ated host defence gene transcription and reduced colonization of a P. microcarpus isolate that provided less N than its competitors. Similarly, the induction of sever al defence-r elated pathways in late sta ge of symbiosis establishment in Populus -L. bicolor ECM mycorrhizal root tips was suggested to be a means of host control to curtail over-colonization by the fungal partner (Plett et al. 2014 ). A secondary reason for the involvement of defence pathways in colonized roots could be due to direct induction of defences by sugar molecules themselves. Se v er al pathogens have been found that manipulate plant hosts to r edir ect sugar into infected tissues (Chen et al. 2010, Chong et al. 2014. The perception of glucose molecules in these situations then leads to induction of innate cell immunity (Baena-Gonzalez 2010 , Rampitsch and Bykova 2012 , Guo et al. 2013 ). Ther efor e, defence-and str ess-r elated gene tr anscription may be corr elated to C presence and transfer at the symbiotic interface, not because these pathways benefit the ECM fungus, but because the plant is pr e v enting C dr aina ge to ECM fungi. T his ma y also pro vide an explanation as to why a negative correlation between C acquisition and colonization rate was observed in our study as the plant host may be activ el y r educing colonization of the more C-demanding fungi. Furthermor e, a positiv e corr elation w as found betw een C acquisition and the depth of the Hartig net, which may suggest that fungi that can penetrate further into the root tip to r eceiv e C are better able to tolerate plant defence mechanisms . T he role of plant defence and str ess mec hanisms in controlling colonization and C acquisition by ECM fungi should be further investigated. Also, future studies on mechanisms behind C transfer in ECM symbioses should take into account that function in mycorrhizal symbioses is highly complex and can be influenced at various le v els other than tr anscriptional r egulation, including pr otein localization and activity.
The exploration type of fungal mycelium, which describes the rate and form of a given ECM fungal species to gr ow thr ough soil (Agerer 2001 ), may drive levels of C acquired from a host. Based on one species, Pisolithus hyphae are classified as a long-distance exploration type, defined as having sparser but further-r eac hing mycelium (Agerer 2001 ). Not only does the current study demonstrate that this may be an over-simplification, as mycelial growth form v aried significantl y between the tested isolates of P. microcarpus, host C in fungal tissues was found not to be positiv el y corr elated with r adial mycelial gr owth r ate or thic kness of the fungal mantle surrounding plant root tips, but rather with density of fungal mycelium and Hartig net depth. This implies that hyphal br anc hing and a dense mycelium growth pattern might be a better predictor of high C acquisition than extension of mycelia into the substrate. Using a microcosm-based study system, Wu et al. ( 2002 ) found similar results whereby ECM fungi with denser hyphal growth concentrated higher portions of host C. This would support the hypothesis that one mechanism by which ECM fungi acquir e C fr om their host is due to a gr eater sink str ength cr eated by fungal biomass, although the signalling connecting growth c har acteristics to C acquisition remain to be investigated in future studies. While such research questions are typically hard to measur e in natur al systems, it is known that host C accum ulates most in areas with high fungal biomass (Leake et al. 2001 ). Furthermore, when C is nonlimiting to the host (e.g. due to ele v ated CO 2 , during summer), ECM species with faster growth and thicker mantles around colonized roots are favoured, while the converse is true when the host is C-limited (e.g. due to defoliation, during winter; Godbold et al. 1997, Markkola et al. 2004, P arr ent and Vilgal ys 2007, Pritc hard et al. 2008, Sar av esi et al. 2008 ). These previous results would argue that, in addition to the findings of this study regarding fungal C demand, the C status of the host is also a driver of photosynthate allocation to ECM partners . T his raises the question of whether a high amount of host-deliv er ed C causes high fungal biomass production or if inherent growth properties of the fungi result in C supply by the host. ECM C transfer is likely to be both a cause and consequence of fungal biomass production, as well as of plant nutrient r equir ements and fungal ability to acquire and deliver these nutrients from soil.
The likelihood of an ECM fungus to form a C sink may depend on its ability not only to acquire C from its host but also to rapidly metabolise C into various intermediate storage compounds, including trehalose and glycogen (López et al. 2007, Wiemken 2007, Nehls 2008. Genomic and transcriptomic studies have indicated the abilities of se v er al ECM fungal species, suc h as L. bicolor , Tuber melanosporum , and A. muscaria , to both utilize sugars and synthesize C stor a ge compounds during symbiosis (De v eau et al. 2008, Nehls 2008, Ceccaroli et al. 2011. Plett et al. ( 2021 ) found that when growing under the same glucose availability as used in the current study (0.01% w/v), P. microcarpus isolates MW3, SI9, R10, and R4 all had little to no glucose in the free sugar compositions of their mycelia, and trehalose was instead the predominant storage sugar. The analysis of fungal C metabolism gene expression in this study r e v ealed that P. microcarpus fungi with significantly different C acquisition values had different expression levels of enzymes for the utilization of sugars, such as fructose and glucose, and for the biosynthesis of the carbohydr ates tr ehalose, gl ycogen, galactinol, and inositol. Ho w e v er, expr ession was not necessaril y higher in the fungi with higher C values and Pearson's correlation analysis of fungal C metabolism genes did not find genes significantl y positiv el y corr elated with C acquisition. It should be noted that neither the study by Plett et al. ( 2021 ) nor the current study examined C acquisition within colonized plant root tips, which are the actual site of C exchange between plants and ECM fungi, but rather ERM. Further work combining the stable isotope and sugar composition analyses from the two studies, to confirm that host-acquired glucose is converted to C storage compounds at the Hartig net within colonized root tips, would pr ovide gr eater understanding of the potential mechanisms of C sink formation by ECM fungi.
Together, the findings of this study suggest that ECM fungal acquisition of host-deriv ed C involv es both fungal and plant mechanisms . T his study pro vides early indications that C acquisition may be highl y v ariable within a given species of ECM fungi, wher eby this tr ait is de pendent on indi vidual C demands as determined by the ability of the fungus to metabolize host-derived C and convert it into biomass. Our study also demonstrates that C acquisition is not pur el y under the control of the fungus, but also depends on the ability of the host plant to reduce colonization and, ther efor e, C dr ain. Adv ances in curr ent understanding of the molecular mechanisms behind the exchange of C within the ECM symbiosis will be essential in better understanding the roles of this plant-fungus association in the C cycling of temperate and bor eal for est ecosystems and, thus, in the global terrestrial C sink.

Ac kno wledgements
We also thank UC Davis Stable Isotope Facility for running the stable isotope analyses and Western Sydne y Uni versity Confocal Bio-Imaging Facility for access to its instrumentation.

Supplementary data
Supplementary data are available at FEMSEC online.

Conflict of inter est.
Authors declar e no conflicts of inter est.